Marine aquaculture in general, and fish farming in particular, have been extensively developed in recent years. While there has been considerable success in achieving high yields in rearing fish, there has been only limited success in the manipulation of the reproductive cycles and spawning of the reared fish. Such manipulation is a prerequisite for the further development of fish farming into a major agricultural industry.
Many of the economically important fish do not reproduce spontaneously in captivity. This is the case with mullet (Mugil cephalus), rabbitfish (Siganus sp.), milkfish (Chanos chanos), striped bass (Morone saxatilis), sea bass (Dicentrarchus labrax), seabream (Sparus aurata), catfish (Clarias sp.) and others. In all these species the reproductive failure is located in the female: whereas vitellogenesis is completed, the stages that follow, namely oocyte maturation and ovulation, do not occur, and thus there is no spawning. Instead, vitellogenic follicles undergo rapid atresia.
In some fish species which do ovulate spontaneously in captivity, such as trout and salmon, both Atlantic and Pacific, e.g. Atlantic salmon (Salmo salar) and Pacific salmon (Onchorhynchus sp.), ovulation is not synchronized and thus egg collection is a very laborious task. Additionally, the subsequent hatching of the fingerlings is not synchronized and therefore the ability to create schools of fingerlings being all at about the same growing stage, which is necessary for economically feasible fish farming, becomes very difficult.
In fish indigenous to temperate zones, such as seabream, seabass, striped bass, cyprinids and salmoneds, reproduction is seasonal, i.e. ovulation and subsequent spawning occur once or several times during a limited season. Inducing such fish to ovulate and spawn out of the natural spawning season might largely contribute to the management of fish farming. For one, out of season egg production may enable full utilization of the fish farm throughout the whole year, by making it possible to have at any given time fish of all ages. Overcoming the restrictions of seasonal spawning, therefore, may enable the marketing of adult fish year round.
Ovulation and spawning in female fish are controlled by pituitary hormones, mainly the gonadotropins (GtH). However, the release of GtH is not spontaneous but rather induced by a gonadotropin releasing hormone (GnRH) which is secreted by the hypothalamus. It has been found in female Sparus aurata, that the level of GtH in the pituitary gland increases as the fish approaches its natural spawning season, i.e. winter time. However, this accumulated GtH is not released into the blood, the consequence being that oocytes undergo rapid atresia. In cases where ovulation and spawning do occur, these are always accompanied by a GtH surge in the blood (Stacey et al., 1979, Gen. Comp. Endocrinol. 27: 246-249; Zohar et al., 1986, Gen. Comp. Endocrinol. 64: 189-198; Zohar et al., 1987, In: Proceedings of the 3rd Int. Symp. on Reprod. Physiol. of Fish. St. John's, Newfoundland, Canada, August 1987). Such a surge of GtH and the subsequent ovulation and spawning may be induced by injection of GnRH or analogs thereof. The use of natural fish GnRH in inducing ovulation and spawning has been described in U.S. Pat. No. 4,443,368 and the use of various analogs thereof has been described in U.K. Published Patent Application No. 2152342 and in U.S. Pat. No. 4,410,514. The use of luteinizing hormone releasing hormones (LHRH) for inducing spawning in fish has been described in Japanese Published Patent Application 80-40210.
The administration of GnRH and the genetic manipulation of its production are therefore excellent candidates for methods of manipulating ovulation and spawning in fish. Methods of administering GnRH and its analogs to fish are described in detail in U.S. Pat. No. 5,288,705 (particularly describing implantation and sustained release), which is incorporated herein by reference in its entirety, and in U.S. Pat. No. 5,076,208 (particularly describing ultrasound mediated uptake in an aquatic medium), which is also incorporated herein by reference in its entirety.
GnRH is a ten amino-acid peptide, synthesized in the hypothalamus and released into the hypophysial portal blood system or directly into the pituitary gland in the case of teleost fish. In the pituitary, GnRH regulates GtH secretion by the gonadotroph cells. Gonadotropins, in turn, stimulate steroidogenesis in the gonads and thereby control oogenesis and spermatogenesis.
Previously, eight forms of GnRH had been isolated from vertebrate brains and characterized. (FIG. 1). They are traditionally named for the species from which they were first isolated. The eight previously known members of the GnRH peptide family are all decapeptides with a highly conserved structure: modified N-terminal (pGlu) and C-terminal (NH.sub.2) residues, and, with the exception of jawless fish GnRHs, conserved amino acids at position 1-4, 6, 9 and 10. For the remaining positions, amino acid 7 is either Trp or Leu and amino acid 5 is either His or Tyr, whereas amino acid 8 was found to be highly variable.
The first GnRH was isolated and characterized from mammals in the early 1970s (Matsuo et al., 1971, Biochem. Biophys. Res. Commun. 43: 1334-1339; Schally et al., 1971, Biochem. Biophys. Res. Commun. 43: 393-399; Amoss et al., 1971, Biochem. Biophys. Res. Commun. 44: 205-210) and is now referred to as mammalian GnRH (mGnRH). mGnRH is the main GnRH form present in mammals and has also been reported to be present in amphibia and a number of primitive bony fishes (Conlon et al., 1993, Endocrinology 132: 2117-2123; King et al., 1990, Peptides 11: 507-514; and Sherwood et al., 1991, Gen. Comp. Endocrinol. 84: 44-57). Two GnRH forms have been isolated from chicken hypothalamus; chicken GnRH-I (cGnRH-I) (King et al., 1982, J. Biol. Chem. 257: 10729-10732) and chicken GnRH-II (cGnRH-II) (Miyamoto et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81: 3874-3878). cGnRH-II apparently is present in all jawed vertebrates except for placental mammals. Another important GnRH variant has been isolated from salmon (sGnRH) (Sherwood et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80: 2794-2798) and was subsequently found to be widely distributed amongst the teleost species. Other species-specific GnRH variants have been and sequenced in catfish (cfGnRH) (Ngamvonchon, et al., 1992, Mol. Cell. Neurosci. 3: 17-22) and dogfish (dfGnRH) (Lovejoy et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89: 6373-6377) and two forms in lamprey; lamprey GnRH-I (Sherwood et al., 1986, J. Biol. Chem. 261: 4812-4819) and lamprey GnRH-III (Sower et al., 1993, Endocrinol. 132: 1125-1131).
The cDNA sequences encoding mGnRH, cGnRH-I, cfGnRH, sGnRH and cGnRH-II precursors have been isolated and characterized from mammals and amphibia, birds and teleost fish (see references in Table 1, and FIG. 9). These cDNAs predict a precursor polypeptide consisting of a leader peptide at the N-terminal in direct linkage with the GnRH decapeptide; followed by a 3 amino acid processing site (Gly-Lys-Arg); and an additional peptide called GnRH associated peptide (GAP). The precursor is processed by cleavage at the dibasic amino acids (Lys-Arg). GnRH and GAP are then stored within the secretory granules until secreted (Wetsel et al., 1991, Endocrinol. 129: 1584-1594).
TABLE 1 Characterized cDNAs of GnRH precursors. GnRH form Species References mGnRH Human Seeburg & Adelman 1984 Nature 31: 666-668 Rat Adelman et al. 1986 Proc. Natl. Acad. Sci. USA 83: 179-183. Mouse Mason et al. 1986 Science 234: 1372-1378. Frog Hayes et al. 1994 Endocrinology 134(4): 1835-1845. cGnRH-I Chicken Dunn et al. 1993 J. Mol. Endocrinol. 11: 19-29. CfGnRH Catfish Bogerd et al. 1994 Eur. J. Biochem. 222(2): 541-549. sGnRH African cichlid Bond et al. 1991 Mol. Endocrinol. 5: 931-932. Rainbow trout Alestrom et al. 1992 Mol. Marine Biol. and Biotech. 1(4/5): 376-379. Atlantic salmon Klungland et al. 1992 Mol. and Cell. Endocrin. 84: 167-174. Masu salmon Suzuki et al. 1992 J. Mol. Endocrin. 9: 73-82. Sockeye Ashihara et al. 1994 Sequence listing salmon copied from a poster which appeared at the IUBS Symposium on "Advances in the Molecular Endocrinology of Fish," May 23-25, 1993, Toronto, Canada. cGnRH-II Catfish Bogerd et al. 1994 African cichlid White et al. 1994 Proc. Natl. Acad. Sci. USA 91: 1423-1427.
Despite the knowledge of these other GnRH's, there remains a need in marine aquaculture for more effective manipulation of reproduction. Obtaining the most physiologically relevant form of the GnRH hormone and its gene will greatly contribute to improving the efficiency of controlling fish reproduction.
The following abbreviations are used in this application in addition to the usual abbreviations for the trivial names of the more common .alpha.-amino acids:
Nva=norvaline PA1 Orn=ornithine PA1 Ile=isoleucine PA1 Nle=norleucine PA1 Nal=.beta.-naphthyl-Ala PA1 Phg=C-phenylglycine PA1 Abu=2-aminobutyric acid PA1 Chg=2-cyclohexyl Gly PA1 OMe=methylester PA1 OBzl=benzyl ester PA1 tBu=tertiary butyl PA1 BOC=tert-butyloxycarbonyl